Fatty acid oxidation is essential for egg production by the parasitic flatworm Schistosoma mansoni

Abstract

Schistosomes, parasitic flatworms that cause the neglected tropical disease schistosomiasis, have been considered to have an entirely carbohydrate based metabolism, with glycolysis playing a dominant role in the adult parasites. However, we have discovered a close link between mitochondrial oxygen consumption by female schistosomes and their ability to produce eggs. We show that oxygen consumption rates (OCR) and egg production are significantly diminished by pharmacologic inhibition of carnitine palmitoyl transferase 1 (CPT1), which catalyzes a rate limiting step in fatty acid β-oxidation (FAO) and by genetic loss of function of acyl CoA synthetase, which complexes with CPT1 and activates long chain FA for use in FAO, and of acyl CoA dehydrogenase, which catalyzes the first step in FAO within mitochondria. Declines in OCR and egg production correlate with changes in a network of lipid droplets within cells in a specialized reproductive organ, the vitellarium. Our data point to the importance of regulated lipid stores and FAO for the compartmentalized process of egg production in schistosomes.

Figure 1. Fecund female schistosomes have high mitochondrial OCR.

A. OCR of Fecund female parasites recovered from mixed sex infections, and Virgin adult females recovered from single sex infections were measured in real time by extracellular flux analysis, at basal (immediately ex-vivo) and following the addition of oligimycin, FCCP and rotenone (Rot) and antimycin A (Ant) at the times indicated. B. Average basal OCR readings of Fecund and Virgin females over the first 30 minutes ex-vivo. C. Spare respiratory capacity of Fecund and Virgin females, calculated as shown in Fig. S1. Data are means plus SEM of readings from 4–5 individual female worms per experiment. Data are representative of at least 3 individual experiments. See also Fig. S1.

Figure 2. OXPHOS is necessary for egg production.

A. Eggs produced per fecund female during the first 24 h ex vivo, in the absence (Ctrl) or presence of oligomycin (Olig), antimycin A (Ant) or rotenone (Rot). B. Survival of females, compared to untreated cultured worms, during the same period and conditionS as described in A. C. Egg production between 24 h and 48 h in vitro following the washing out of inhibitors that were present during the first 24 h ex vivo. Data are means plus SEM of readings from 10 individual female worms per experiment. Data are representative of at least 3 individual experiments.

Figure 3. Schistosomes use FA from lipid droplets for FAO to produce eggs.

A. Average basal OCR of fecund females incubated without (Ctrl) or with etomoxir (ETO) over the first 30 min ex vivo. See also Fig. S2. B. Numbers of eggs produced in 24 h per female parasite in the absence or presence of etomoxir. C Oil-Red-O stained fecund females immediately ex vivo or at day 3 or day 13 of culture (red = Oil Red O; blue = Hoescht; green = phalloidin) and quantitation of Oil-Red-O staining of females, as indicated. Images are optical sections through longitudinal axes. Scale bar = 50 µm. D. Palmitate induced mitochondrial FAO (% basal OCR) of fecund females ex vivo and after 13 days in culture. E. Average basal OCR of fecund females ex vivo and after 3 or 13 days in culture (black bars) and numbers of eggs produced within the 24 h period immediately ex vivo or in the 24 h period prior to day 3 or day 13 of culture (pink circles). F. Quantitation of Oil-Red-O staining of fecund females cultured without or with etomoxir for 24 h. Data are means plus SEM of readings from 5–6 individual female worms per experiment. Data are representative of at least 3 individual experiments. ns = not significant.

Figure 4. Loss of ACSL and ACAD function inhibits egg production.

A. Average basal OCR of fecund females incubated without (Ctrl) or with Triascin C (TC) over the first 30 min ex vivo. B. Numbers of eggs produced in 24 h per female parasite in the absence or presence of Triascin C. Average basal OCR (C & F), numbers of eggs produced in 72 h per female (D & G) and quantitation of Oil-Red-O staining (E) and measurement of FAO activity (H) in control fecund females, and in fecund females electroporated with SmASCL-siRNA (siASCL) or SmACAD-siRNA (siACAD), or with control siRNA (-ve siRNA). Data are means plus SEM of readings from 5–6 individual female worms per experiment. Data are representative of at least 2 individual experiments. See also Fig. S2.